Method for delivering benzindene prostaglandins by inhalation

ABSTRACT

A method of delivering benzindene prostaglandins to a patient by inhalation is discussed. A benzindene prostaglandin known as UT-15 has unexpectedly superior results when administered by inhalation compared to parenterally administered UT-15 in sheep with induced pulmonary hypertension.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/525,471, filed on Mar. 15, 2000, now U.S. Pat. No. 6,521,212 whichclaims priority to provisional U.S. Application Serial No. 60/124,999,filed Mar. 18, 1999.

BACKGROUND OF THE INVENTION

Benzindene prostaglandins are now known to be useful to treat a varietyof conditions. U.S. Pat. No. 5,153,222 describes the use of a preferredclass of benzindene prostaglandins in the treatment of pulmonaryhypertension, including both primary and secondary pulmonaryhypertension. In particular, this patent discusses the use of thecompound compound9-deoxy-2′,9-alpha-methano-3-oxa-4,5,6-trinor-3,7-(1′,3′-interphenylene)-13,14-dihydro-prostaglandinF₁ (also known as UT-15).

However, this patent does not specifically suggest the administration ofsuch benzindene prostaglandins by inhalation or the surprising benefitsthat result from their delivery by inhalation.

U.S. Pat. No. 4,306,075 describes a large group of carbacyclin analogs,including benzindene prostaglandins, which produce variouspharmacological responses, such as inhibition of platelet aggregation,reduction of gastric secretion, and bronchodilation. It is indicatedthat the compounds have useful application as anti-thrombotic agents,anti-hypertension agents, anti-ulcer agents, and anti-asthma agents. Thepatent does mention administration by inhalation. The patentspecifically discloses the compound UT-15 in Example 33. However, thispatent provides only limited biological data relating to the use of suchcompounds. At column 59, example 31, the patent discloses a compoundthat is structurally similar to that of example 33 (UT-15), but it isnot the same compound. Example 31 discloses (column 59, lines 41-45)that “[t]he compounds [sic]9-deoxy-2′,9α-methano-3-oxa-4,5,6-trinor-3,7-(1′,3′-interphenylene)-PGF₁,methyl ester, given to a rat orally at a dose of 1 mg/kg lowered bloodpressure 44 mmHg. After 52 min the blood pressure was still lower 14mm.”

All blood is driven through the lungs via the pulmonary circulation inorder, among other things, to replenish the oxygen which it dispenses inits passage around the rest of the body via the systemic circulation.The flow through both circulations is in normal circumstances equal, butthe resistance offered to it in the pulmonary circulation is generallymuch less than that of the systemic circulation. When the resistance topulmonary blood flow increases, the pressure in the circulation isgreater for any particular flow. This is referred to as pulmonaryhypertension. Generally, pulmonary hypertension is defined throughobservations of pressures above the normal range pertaining in themajority of people residing at the same altitude and engaged in similaractivities.

Most often pulmonary hypertension is a manifestation of an obvious orexplicable increase in resistance, such as obstruction to blood flow bypulmonary emboli, malfunction of the heart's valves or muscle inhandling blood after its passage through the lungs, diminution inpulmonary vessel caliber as a reflex response to hypoventilation and lowoxygenation, or a mismatch of vascular capacity and essential bloodflow, such as shunting of blood in congenital abnormalities or surgicalremoval of lung tissue. Such pulmonary hypertension is referred to assecondary hypertension.

There remain some cases of pulmonary hypertension where the cause of theincreased resistance is as yet inexplicable. They are described asprimary pulmonary hypertension (PPH) and are diagnosed by and afterexclusion of the causes of secondary pulmonary hypertension. Despite thepossibility of a varied etiology, cases of primary pulmonaryhypertension tend to comprise a recognizable entity. Approximately 65%are female and young adults are most commonly afflicted, though it hasoccurred in children and patients over 50. Life expectancy from the timeof diagnosis is short, about 3 to 5 years, though occasional reports ofspontaneous remission and longer survival are to be expected given thenature of the diagnostic process. Generally, however, progress isinexorable via syncope and right heart failure and death is quite oftensudden.

Pulmonary hypertension refers to a condition associated with anelevation of pulmonary arterial pressure (PAP) over normal levels. Inhumans, a typical mean PAP is approximately 12-15 mm Hg. Pulmonaryhypertension, on the other hand, is sometimes marked by PAP increases byat least 5 to 10 mm Hg over normal levels. PAP readings as high as 50 to100 mm Hg over normal levels have been reported. When the PAP markedlyincreases, plasma can escape from the capillaries into the lunginterstitium and alveoli. Fluid buildup in the lung (pulmonary edema)can result, with an associated decrease in lung function that can insome cases be fatal.

Pulmonary hypertension may either be acute or chronic. Acute pulmonaryhypertension is often a potentially reversible phenomenon generallyattributable to constriction of the smooth muscle of the pulmonary bloodvessels, which may be triggered by such conditions as hypoxia (as inhigh-altitude sickness), acidosis, inflammation, or pulmonary embolism.Chronic pulmonary hypertension is characterized by major structuralchanges in the pulmonary vasculature, which result in a decreasedcross-sectional area of the pulmonary blood vessels. This may be causedby, for example, chronic hypoxia, thromboembolism, or unknown causes(idiopathic or primary pulmonary hypertension).

Pulmonary hypertension has been implicated in several life-threateningclinical conditions, such as adult respiratory distress syndrome(“ARDS”) and persistent pulmonary hypertension of the newborn (“PPHN”).Zapol et al., Acute Respiratory Failure, p. 241-273, Marcel Dekker, NewYork (1985); Peckham, J. Ped. 93:1005 (1978). PPHN, a disorder thatprimarily affects full-term infants, is characterized by elevatedpulmonary vascular resistance, pulmonary arterial hypertension, andright-to-left shunting of blood through the patent ductus arteriosus andforamen ovale of the newborn's heart. Mortality rates range from 12-50%.Fox, Pediatrics 59:205 (1977); Dworetz, Pediatrics 84:1 (1989).Pulmonary hypertension may also result in a potentially fatal heartcondition known as “cor pulmonale”, or pulmonary heart disease. Fishman,“Pulmonary Diseases and Disorders” 2^(nd) Ed., McGraw-Hill, N.Y. (1988).

The treatment of pulmonary hypertension by the parenteral administrationof certain prostaglandin endoperoxides, such as prostacyclin (also knownas flolan), is also known and is the subject of U.S. Pat. No. 4,883,812.Prostacyclin has been administered by inhalation and is used to treatpulmonary hypertension by inhalation. Anesthesiology, vol. 82, no. 6,pp. 1315-1317.

SUMMARY OF THE INVENTION

This invention relates to the administration of a therapeuticallyeffective amount of a benzindene prostaglandin to a mammal in needthereof by inhalation. More particularly, the invention relates to amethod of treating pulmonary hypertension by administering an effectiveamount of a benzindene prostaglandin to a mammal in need thereof byinhalation.

Inhalation of benzindene prostaglandins provides unexpectedly superiorresults compared to parenteral administration of benzindeneprostaglandins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of pulmonary vascular resistance (cmH₂O*min/liter)intravenously induced by U44069 over time (min).

FIG. 2 describes the effects of a high dose of UT15, given as anaerosol, on the hemodynamic variables of the sheep. Specifically, FIG. 2depicts the effects of the aerosolized UT15 administered to the sheepintravenously induced with U44069 on systemic arterial pressure(PSA orPSYS); on pulmonary arterial pressure (PPA); and pulmonary vascularresistance (PVR), respectively.

FIG. 3 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on the heart rate during baseline conditions.

FIG. 4 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on the systemic arterial pressure during baselineconditions.

FIG. 5 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on the central venous pressure during baselineconditions.

FIG. 6 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on the pulmonary arterial pressure during baselineconditions.

FIG. 7 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on the left atrial pressure during baseline conditions.

FIG. 8 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on cardiac output during baseline conditions.

FIG. 9 is the dose-response effect of intravenously infused UT15 andaerosolized UT15 on pulmonary vascular resistance during baselineconditions.

FIG. 10 is the dose-response effect on the heart rate of intravenouslyinfused UT15 and aerosolized UT15 during intravenously infused U44069.

FIG. 11 is the dose-response effect of intravenously infused andaerosolized UT15 on central venous pressure during intravenously infusedU44069.

FIG. 12 is the dose-response effect of intravenously infused andaerosolized UT15 on systemic arterial pressure during intravenouslyinfused U44069.

FIG. 13 is the dose-response effect of intravenously infused andaerosolized UT15 on pulmonary arterial pressure during intravenouslyinfused U44069.

FIG. 14 is the dose-response effect of intravenously infused andaerosolized UT15 on left atrial pressure during intravenously infusedU44069.

FIG. 15 is the dose-response effect of intravenously infused andaerosolized UT15 on cardiac output during intravenously infused U44069.

FIG. 16 is the dose-response effect of intravenously infused andaerosolized UT15 on pulmonary vascular resistance during intravenouslyinfused U44069.

FIG. 17 is the dose-response effect of intravenously infused andaerosolized UT15 on pulmonary vascular driving pressure (PPA minus PLA)during baseline conditions.

FIG. 18 is the dose-response effect of intravenously infused andaerosolized UT15 on pulmonary vascular driving pressure (PPA-PLA) duringintravenously infused U44069.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, all references to “a” or “an” mean at leastone.

One embodiment of the present invention is a method of delivering abenzindene prostaglandin or a pharmaceutically acceptable salt or esterthereof to a mammal in need thereof by inhalation.

A preferred group of benzindene prostaglandins for delivery byinhalation according to the present invention is as follows:

wherein a is an integer of from 1 to 3; X and Y, which may be the sameor different, are selected from —O— and —CH₂—; R is —(CH₂)₅—R¹ whereinR¹ is hydrogen or methyl, or R is cyclohexyl, or R is —CH(CH₃)CH₂C CCH₃;and the dotted line represents an optional double bond; or aphysiologically acceptable salt or acid derivative thereof.

The most preferred benzindene prostaglandin is UT-15, which is9-deoxy-2′,9-alpha-methano-3-oxa-4,5,6-trinor-3,7-(1′,3′-interphenylene)-13,14-dihydro-prostaglandinF₁.

“Inhalation” delivery in the context of this invention refers to thedelivery of the active ingredient or combination of active ingredientsthrough a respiratory passage, wherein the mammal in need of the activeingredient(s) inhales the active ingredient(s) through the mammal'sairways, such as the nose or mouth.

Active ingredients, which are aerosolized, atomized, and/or nebulizedfor delivery by inhalation according to the present invention includeliquid formulations comprising a benzindene prostaglandin, such asUT-15, alone or in combination with other active ingredients describedbelow. UT-15 may be used as a free acid or in the form of apharmaceutically acceptable salt or ester or other acid derivative. Inaddition, sustained release formulations comprising UT-15 may be used,including PEGylated forms and/or protein-conjugated forms of UT-15.

The term “acid derivative” is used herein to describe C, alkyl estersand amides, including amides wherein the nitrogen is optionallysubstituted by one or two C₁₋₄ alkyl groups.

The invention also includes bioprecursors or “pro-drugs” of UT-15, thatis, compounds which are converted in vivo to UT-15 or itspharmaceutically active derivatives thereof.

Further aspects of the present invention are concerned with the use ofUT-15, or a pharmaceutically acceptable salt or acid derivative thereof,in the manufacture of a medicament for the treatment of peripheralvascular disease

The present invention extends to non-physiologically acceptable salts ofUT-15 which may be used in the preparation of the pharmacologicallyactive compounds of the invention. The physiologically acceptable saltsof UT-15 include salts derived from bases.

Base salts include ammonium salts, alkali metal salts such as those ofsodium and potassium, alkaline earth metal salts such as those ofcalcium and magnesium, salts with organic bases such asdicyclohexylamine and N-methyl-D-glucamine, and salts with amino acidssuch as arginine and lysine.

Quaternary ammonium salts can be formed, for example, by reaction withlower alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides,bromides, and iodides, with dialkyl sulphates, with long chain halides,such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, andiodides, and with aralkyl halides, such as benzyl and phenethylbromides.

Optionally, one or more pharmaceutically acceptable carriers orexcipients may be included in the formulation to be aerosolized,atomized, or nebulized according to the invention.

A preferred solution for administration by inhalation with a nebulizerincludes a sterile solution of UT-15 comprising UT-15, sodium citrate,citric acid, sodium hydroxide, sodium chloride, and meta-cresol. A morepreferred solution is prepared by mixing 0.125 grams UT-15, 1.25 gramshydrous sodium citrate, 0.125 grams of anhydrous citric acid, 0.05 gramsof sodium hydroxide, and approximately 250 ml of water for injection.

Preferably, a nebulizer, inhaler, atomizer or aerosolizer is used whichforms droplets from a solution or liquid containing the activeingredient(s). The droplets are preferably less than 10 micrometers indiameter. One preferred nebulizer is the AM-601 MEDICATOR AEROSOLDELIVERY SYSTEM™ (a nebulizer manufactured by Healthline Medical inBaldwin Park, Calif.).

Alternatively, solid formulations, usually in the form of a powder, maybe inhaled in accordance with the present invention. In such case, theparticles are preferably less than 10 micrometers in diameter, and morepreferably, less than 5 micrometers in diameter.

This invention further relates to delivering a benzindene prostaglandinand/or its salts pr esters by inhalation for applications whereinhalation delivery is appropriate for the treatment of that particularcondition. Benzindene prostaglandins, including UT-15 and its salts oresters, have been shown to be useful for multiple applications. Forexample, UT-15 has been shown to exhibit a potent anti-aggregatoryaction on blood platelets, and therefore has a particular utility inmammals as an anti-thrombotic agent. Further known uses of UT-15 includetreatments of pheripheral vascular disease (covered in co-pendingapplication Ser. No. 09/190,450, now U.S. Pat. No. 6,054,486, the entirecontents of which are incorporated by reference herein). In the case oftreating peripheral vascular disease by inhalation of a benzindeneprostaglandin of the present invention, the dosage for inhalation,taking into account that some of the active ingredient is breathed outand not taken into the bloodstream, should be sufficient to deliver anamount that is equivalent to a daily infusion dose in the range of 25 μgto 250 mg; typically from 0.5 μg to 2.5 mg, preferably from 7 μg to 285μg, per day per kilogram bodyweight. For example, an intravenous dose inthe range 0.5 μg to 1.5 mg per kilogram bodyweight per day mayconveniently be administered as an infusion of from 0.5 ng to 1.0 μg perkilogram bodyweight per minute. A preferred dosage is 10 ng/kg/min.

Benzindene prostaglandins, including UT-15 and its salts or esters, mayalso be administered according to the present invention by inhalation toreduce and control excessive gastric secretion, thereby reducing oravoiding gastrointestinal ulcer formation, and accelerating the healingof ulcers and lesions already present in the gastrointestinal tract. Inaddition, benzindene prostaglandins may also be administered accordingto the present invention by inhalation to treat congestive heartfailure, to reduce inflammation and/or pulmonary hypertension associatedwith lung transplants.

Benzindene prostaglandins, including UT-15 and its salts or esters,further exhibit vasodilatory action on blood vessels and therefore havea particular utility as anti-hypertensives for the treatment of highblood pressure in mammals, including man. Use as an anti-hypertensive(or hypotensive agent) may be accomplished by administering apharmaceutical composition containing a benzindene prostaglandin,including UT-15.

Benzindene prostaglandins, including UT-15, may be used according to thepresent invention by inhalation to treat any condition where it isdesired to reduce blood pressure, inhibit platelet aggregation, toreduce the adhesive character of platelets, and/or to treat or preventthe formation of thrombi in mammals, including man. For example, theymay be used in the treatment and prevention of myocardial infarcts andin the treatment of peripheral vascular disease, to treat and preventpost-operative thrombosis, to promote patency of vascular graftsfollowing surgery, and to treat complications of arteriosclerosis andconditions such as atherosclerosis, blood clotting defects due tolipemia, and other clinical conditions in which the underlying etiologyis associated with lipid imbalance or hyperlipidemia. Moreover,benzindene prostaglandins, including UT-15 and its salts or esters, havea further utility in the promotion of wound healing in mammals,including man.

Benzindene prostaglandins, including UT-15 and its salts or esters, mayalso be used as additives to blood, blood products, blood substitutes,and other fluids, which are used in artificial extra-corporealcirculation and perfusion of isolated body portions, e.g., limbs andorgans, whether attached to the original body, detached and beingpreserved or prepared for transplant, or attached to a new body. Duringthese circulations and perfusions, aggregated platelets tend to blockthe blood vessels and portions of the circulation apparatus. Thisblocking is avoided by the presence of UT-15. For this purpose, UT-15 orits salts or esters may be introduced by inhalation until it reaches alevel in the circulating blood, the blood of the donor animal, or theblood of the perfused body portion, or to two or all of those equivalentto a steady state dose of 0.001 micrograms to 10 micrograms, per literof circulating fluid. Another embodiment is to use UT-15 in laboratoryanimals, e.g., cats, dogs, rabbits, monkeys and rats, for these purposesin order to develop new methods and techniques for organ and limbtransplants.

In accordance with the present invention, a benzindene prostaglandin isdelivered by inhalation to a patient in need thereof in a“therapeutically effective amount”. A “therapeutically effective amount”refers to that amount that has therapeutic effects on the conditionintended to be treated or prevented. For example, an “antihypertensiveeffective amount” refers to that amount in which the effects frompulmonary hypertension, and particularly, pulmonary arterial pressure(PAP), are reduced towards a normal level relative to hypertensivelevels, or maintained at normal levels. The precise amount that isconsidered effective for a particular therapeutic purpose will, ofcourse, depend upon the specific circumstances of the patient beingtreated and the magnitude of effect desired by the patient's doctor.Titration to effect may be used to determine proper dosage.

Such formulations, both for veterinary and for human medical use, of thepresent invention comprise the active ingredient, a benzindeneprostaglandin or salt or ester thereof, together with one or morepharmacologically acceptable carriers therefor and optionally othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof.

Furthermore, the formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. All methods include the step of bringing intoassociation the active ingredient with the carrier which constitutes oneor more pharmacologically acceptable accessory ingredients.

The invention further relates to a method of treating pulmonaryhypertension by inhalation of a benzindene prostaglandin. “Pulmonaryhypertension” refers to both acute and chronic hypertension, includingprimary pulmonary hypertension and secondary pulmonary hypertension, andis associated with an elevated pulmonary arterial pressure over normallevels.

The efficacy of benzindene prostaglandins, such as UT-15, for treatingpulmonary hypertension can be assessed by determining the hemodynamicsassociated with pulmonary hypertension. In particular, measurements ofpulmonary arterial pressure (PPA), left atrial pressure (PLA), centralvenous pressure (PCV), systemic arterial pressure (PSYS), heart rate(HR), and cardiac output (CO) are useful in determining the effects ofbenzindene prostaglandins delivered by inhalation or parenterally.

Although pulmonary arterial pressure can be directly measured and isoften used to quantify pulmonary arterial hypertension, PPA can beaffected by 3 other variables: CO, PLA and PVR, as indicated by Equation1:

PPA=(CO * PVR)+PLA  (1)

As can be seen from Equation 1, PPA can be elevated by increases in PLA(e.g., left heart failure, mitral valave stenosis, mitral valveregurgitation), increases in CO (e.g., low hematocrit, peripheralvasodilation, left to right shunt, etc.), and by increases in PVR(decreased pulmonary vascular surface area, decreased pulmonary vascularradii, pulmonary vascular obstructions, etc.).

On the other hand, PVR can not be directly measured and must becalculated by the following Equation 2:

PVR=(PPA−PLA)/CO  (2)

PVR is a better index of pulmonary arterial hypertension (PAH), sinceinterventions used to treat PAH are best if they only affect PVR andhave no or little effect on CO and PLA.

Heart rate was determined by measuring the time (seconds) required for25 heart beats to occur (t₂₅) as indicated by the pulsations on theblood flow meter; the beats per minute (BPM) were calculated by thefollowing equation:

BPM=(25 beats/t ₂₅) * 60 seconds

All pressure may be monitored by commercially available transducers,such as Model 1290A HEWLETT PACKARD™ transducer (Andover, Mass.), whichis attached to VALIDYNE CD19A Carrier Dmod. Amplifiers (Northridge,Calif.). Cardiac output may be measured by a Transonic Systems T101Ultrasonic Bloodflow Meter (Ithaca, N.Y.). The pressure and blood flowsignals may be recorded on ASTROMED MT-9500 Stripchart Recorder (WestWarwick, R.I.) and digitally recorded with a personal computer usingEasy Data Acquisition Software (Nashville, Tenn.).

It has been discovered that aerosolized UT-15 has both greater potencyand efficacy relative to attenuating chemically induced pulmonaryhypertension as shown by an increase in pulmonary vascular resistance.Furthermore, aerosolized UT-15 has a greater potency as compared tointravascularly administered UT-15, since the actual amount of UT-15delivered via aerosolization delivery is only a fraction (10-50%) of thedosage delivered intravascularly. While the mechanism(s) that accountsfor the greater potency and efficacy for aerosolized UT-15 is unknown,it can be hypothesized that a low “first-pass” uptake via intravenousinfusion of UT-15 could be at least partially responsible. A lowfirst-pass uptake would thus allow the majority of the drug to be madeavailable to the peripheral circulation (including the coronarycirculation), which would increase the heart rate and cardiac output.

Aerosolized UT-15 has no apparent peripheral effects, such as on theheart rate or cardiac output, as compared to intravascular UT-15 duringpulmonary vascular hypertension by chemical inducement. This isparticularly beneficial for those patients that are near right heartfailure and where peripheral vasodilation would exacerbate the challengeto the right heart. On the other hand, if cardiac output is compromiseddue to right heart failure, then aerosolized prostaglandin woulddecrease PVR and could allow cardiac output to increase while allowinglowering the load upon the right heart.

The following examples are provided by way of an illustration of thepresent invention and should in no way be construed as constituting alimitation thereof.

EXAMPLES Example I

Animal Model

Inhalation solutions were prepared by combining 1.25 grams of SodiumCitrate (Hydrous), 0.125 Citric Acid (Anhydrous), 0.05 grams of SodiumHydroxide (NF/BP), 0.125 grams of UT-15, and approximately 250 ml ofWater for Injection according to the following steps.

1. Measured approximately 210 ml of water into a sterile siliconizedglass beaker with a magnetic stir bar

2. Added sodium citrate. Mixed until dissolved.

3. Added citric acid to Step 2 solution. Mixed until dissolved.

4. Measured 12.5 ml of water into sterile plastic tube. Added sodiumhydroxide. Mixed until dissolved.

5. Added UT 15 to Step 4 solution. Mixed by hand until dissolved.

6. Added the Step 5 solution to Step 3 solution and mixed.

7. pH was adjusted using hydrochloric acid and/or sodium hydroxidesolutions to a value of 7.3

8. Final solution was filtered using sterile microfilter into anothersterile beaker, then 5 ml of solution was aliquoted to sterile stopperedblood test tubes.

9. Solutions were double boxed and put in −4 degrees Celsius freezer.

10. Placebo solution made up as described above except UT-15 not addedand quantities adjusted to make only 50 ml.

Working solution was made by adding sterile saline to dilute the UT15stock solution or placebo to the desired amount (depending on dosedesired, weight of sheep, and duration of aerosolizing). This solutionwas then added to the nebulizer in volumes not exceeding 5 ml untilentire amount was used.

For a 35 kg sheep at a UT-15 dose of 250 ng per kg per minute for 30minutes, the calculations used were, Calculations: 250×35×30=262,500 ngof UT-15 or 262.5 micrograms of UT-15. The nebulization rate was 0.28 mlper minute, thus 8.4 ml of solution was needed containing 262.5micrograms of UT-15. However, an amount of solution is needed for the“void” volume (volume always left in the nubulizer). Thus a volume of 9ml containing a total of 281.25 micrograms of UT-15 (or 0.5625 ml of thestock solution) was made up.

0.5625 ml of UT-15 was measured and added to 8.4375 of sterile saline.This was nebulized over exactly 30 minutes.

Sheep were used as the animal model of choice for these experiments fora number of reasons. First is the docile nature of sheep. They willstand quietly in metabolic cages without having to utilize tranquilizingdrugs, which have the potential to complicate experimental results.Second, sheep are large enough to allow direct measurement of CO, PPA,PLA, PCV, and PSYS. Sheep are also large enough to allow directaerosolization of substances into the lung via trachoestomy therebypreventing swallowing of drugs and thus eliminating a possible secondaryroute of administration of UT-15. Third, sheep can tolerate a greatamount of instrumentation with little or no discomfort. Fourth, sheephave been utilized for several years as an animal model of pulmonaryarterial hypertension and thus, there is a great amount of historicaldata with which to compare the results. The agent chosen to inducepulmonary arterial hypertension was a PGH2 analog, U44069(9,11-dideoxy,9α,11α-epoxymethanoprostaglandin F_(2α)). The reasons forusing U44069 are that it is a very potent pulmonary vasoconstrictor, itscharacteristics are very similar to endogenously formed thromboxane A2,and it can be titrated to induce the desired degree of pulmonaryvasoconstriction. U44069 was mixed with sterile normal salineimmediately prior to being used and was protected from light by wrappingthe solution with aluminum foil. The concentration of U44069 wasadjusted such that a minimal flow rate of 0.8 ml per min was beinginfused into the sheep. This was done because more concentrated U44069would have to be infused at very low rates and often causes “pulses” ofU44069 due to the infusion characteristics of roller pumps. The U44069pulses cause vasoconstriction “spikes” and thus would create induce anon-steady-state.

Surgical Procedures

Six yearling sheep (3 males, 3 females; 21-37 kg) were fasted 18-24hours and initially anesthetized with a short acting barbiturate(thiopental) to allow intubation of the sheep. Halothane gas anesthesia(1.5-2.5%) was then used for the surgical procedures. Via a leftthoracotomy, a Transonic blood flow probe was placed around the mainpulmonary artery, silastic catheters placed in the main pulmonary arteryand left atrium. After approximately 7 days the sheep werereanesthetized and the left carotid artery cannulated, a CordisIntroducer Sheath inserted in the left jugular vein, and a tracheotomymade. The sheep were allowed to recover for another 3-5 days prior toexperimentation. These sheep were used to allow measurement of pulmonaryarterial pressure (PPA), left atrial pressure (PLA), central venouspressure (PCV), systemic arterial pressure (PSYS), heart rate (HR), andcardiac output (CO) after baseline measurements were made for a minimumof 30 minutes.

Example II Effects of Prolonged U44069 Intravenous Infusion on PulmonaryVascular Resistance

In four sheep, the ability of U44069 to maintain a steady-state increasein PVR was determined. After a 30 minute baseline, U44069 was infused ata rate of 1 microgram per kg of body weight per minute for 180 minutes.As can be seen by FIG. 1, the increase in PVR induced by intravenouslyinduced U44069 is very stable over 3 hours. (In the figures, all dataare given as mean ±SEM. “*” indicates significantly different fromcorresponding intravenously infusion UT-15 delivery rate. “#” indicatessignificantly different from corresponding baseline value. “&” indicatessignificantly different from corresponding U44069 value.) Statisticalanalysis was also tested using multiple paired t-tests, which are not asrigorous as One-way ANOVA/Dunnett's test. In particular, FIG. 1illustrates that intravenously infused U44069 causes PVR to reach asteady-state increase by 30 minutes and that the steady-state increaselasts for a minimum of 180 minutes. U44069 caused significantalterations in other variables (data not shown) over the 180 minuteinfusion period relative to their baseline values: PPA increased, HRdecreased, CO decreased. PSYS increased above baseline values, however,the differences were not statistically different except at 120, 150 and180 minutes during U44069 infusion. PCV also increased during U44069infusion, however, the increases were only significant at 30 and 60minutes. PLA did not significantly change at any of the time pointsinvestigated.

Since all of the U44069 time values were different from baseline yetnone were different from each other as determined by the paired t-tests,this would argue strongly that there were no differences at any of thetime points during U44069 infusion. These data would indicate that anyalterations in PVR by UT-15 is due to the effects of UT-15 and notcomplicated by waning of the vasoconstrictor response.

Example III Effects of Aerosolized UT-15 Given at High Doses on BaselineHemodynamics

Baseline measurements consisted of 30 minutes of monitoring duringvehicle/saline aerosolization (0.28 ml/min). After baselinemeasurements, the vehicle/saline solution in the aerosol delivery systemwas replaced with the stock UT-15 solution (500 ng/ml) and wasaerosolized at 0.28 ml/min for 90 minutes.

FIG. 2 depicts the only statistically altered variables observed after90 minutes of high dose aerosolized UT-15 (3800-5700 ng per kg per min).PSYS decreased by 7.5%, PPA decreased by approximately 18%, and PVRdecreased by approximately 19% relative to their respective baselinevalues.

These data are important in that this would indicate that, unlikeintravenously infused UT-15, aerosolized UT-15 can be given in highdoses without significant non-lung effects, i.e., heart rate, cardiacoutput. The aerosol delivery of UT-15 for these experiments isapproximately 15-27 times that of the effective minimal tested dose of250 ng per kg per min shown in FIG. 16.

Example IV Control Intravenous UT-15 and Control Aerosolized UT-15 DoseResponse Effects on Baseline Hemodynamics

Two separate experiments were conducted to determine the dose responseeffects of intravenously infused UT-15 on baseline hemodynamics andaerosolized UT-15 on baseline hemodynamics. For the infusionexperimental protocol, after a 30 minute baseline was established, UT-15was infused intravenously at 3 rates (250, 500 and 1000 ng per kg permin). In three sheep, the infusion rates lasted for 30 minutes each, andfor the other three sheep, the infusions were for 60 minutes each.

The aerosolized UT-15 protocol involved establishing a 30 minutebaseline, then administering aerosolized UT-15 via a tracheostromy atrates of 250, 500 and 1000 microgram per kg of body weight per min andat an aerosolization rate of 0.28 ml/min. Again, three sheep wereaerosolized for 30 minutes and the other three sheep were aerosolizedfor 60 minutes.

No differences were found between 30 minute and 60 minute UT-15 deliveryat each of the 3 rates of administration. FIG. 3 shows the dose-responseof intravenously infused and aerosolized UT-15 on heart rate. Heart ratesignificantly increased during intravenous administration of UT-15 at250, 500 and 1000 ng per kg per min. Aerosolized UT15 had no effect onheart rate. There was a significant difference between aerosolized andintravenously infused UT-15 at each of the 3 rates of administration.

FIG. 4 shows that both aerosolized and intravenous UT-15 had nosignificant effect on PSYS at any of the administration rates used.

The effects of UT-15 on PCV are depicted by FIG. 5. There were nostatistical difference at any dose relative to its baseline value norbetween intravenous and aerosol administered UT-15 at any respectivedose. The same effects were also observed for PPA as indicated by FIG.6, although there was a general trend for PPA to decrease when UT-15 wasaerosolized.

Interestingly, while neither intravenous nor aerosolized UT-15 causedPLA to significantly change from their respective baselines (althoughthe mean values increased during aerosol delivery and decreased forintravenous delivery), there were significant differences betweenaerosolized and intravenous administered UT-15 at each of the deliveryrates. See FIG. 7.

FIG. 8 depicts the effects on CO: no significant changes were observedfor any delivery rate relative to the respective baseline values norwere any significant changes observed between the two modes of drugdelivery.

FIG. 9 represents the overall effect of aerosolized and intravenouslyinfused UT-15 on the pulmonary circulation, PVR. Intravenous UT-15 hadno significant effect on PVR whereas aerosolized UT-15 did cause asignificant decrease at all 3 delivery rates.

The decrease in PVR for aerosolized UT-15 at 250, 500, and 100 ng per kgper min is attributable to the small increase in PLA and small decreasein PPA. While neither of these variables were significantly differentfrom the baseline values, the combinations (i.e., PPA minus PLA, used inEquation 2) were significant, as depicted in FIG. 17. Intravascularlyinfused UT-15 had no effect on PVR yet did have significant effects onheart rates. The statistical analysis of these data were done usingrigorous two-way ANOVA and Student-Newman-Keuls tests, thus anystatistical differences can be accepted with confidence.

Example V Constricted Intravenous and Aerosolized UT-15 Dose Response

Two separate experiments were conducted to determine the dose responseeffects of intravenously infused UT-15 and aerosolized UT-15 duringU44069 induced pulmonary hypertension. After a 30 minute baseline wasestablished, U44069 was infused intravenously at a rate of 1 ng per kgper min. For the intravenous administration of UT-15 and after allowingthe sheep to achieve a steady-state for 30-60 minutes, a dose-responseto intravenous UT-15 was similar to that set forth in Example IV. Forthe aerosolized administration of UT-15 and after allowing the sheep toachieve a steady-state for 30-60 minutes, a dose-response to intravenousUT-15 was similar to that set forth in Example IV. In each experimentalprotocol, UT-15 was administered to three sheep for 30 minutes and tothe other three sheep for 60 minutes.

No differences were found between 30 minute and 60 minute UT-15 deliveryat each of the three rates of administration. The effects of U44069 andthe subsequent dose-response effects of UT-15 during U44069 infusion onheart rate are shown in FIG. 10. Intravenous UT-15 caused heart rate toincrease above the values during U44069 conditions, whereas aerosolizedUT-15 had no effect on heart rate. In particular, for intravenous UT-15,the heart rate was significantly different relative to the baseline onlyat a delivery rate of 1000 ng per kg per min, whereas both 500 and 1000ng per kg per min intravenous delivery of UT-15 were statisticallydifferent from the U44069 values. Both 500 and 1000 ng per kg per minaerosol delivery rates were different from their correspondingintravenous infusion delivery rates.

Data for central venous pressure are shown by FIG. 11. Some differenceswere noted for central venous pressure for intravenous UT-15, in that,at 500 and 1000 ng per kg per min delivery rates the values weredifferent from the U44069 values. Only the 500 ng per kg per min aerosolvalue was different from the corresponding intravenous UT-15 infusionvalue.

There were no statistical differences for the systemic arterial pressurefor these series of experiments (FIG. 122). Pulmonary arterial pressureresponses are illustrated by FIG. 13. U44069 significantly increased PPArelative baseline and all 3 delivery rates for significantly greater foraerosolized UT-15 for all 3 rates of drug delivery relative tointravenous delivery. In fact, for aerosolized UT 15 at 500 and 1000 ngper kg per min PPA was back to normal values.

U44069 did not alter left atrial pressure significantly. However,intravenously infused UT-15 caused a significant decrease from theU44069 value at all three delivery rates and were different from thebaseline values at 500 and 1000 ng per kg per min. All three aerosoldelivery rates were increased above baseline, while 250 and 500 ng perkg per min were increased above the U44069 values. As can be seen fromFIG. 14, all three aerosol delivery rate effects were different from theintravenously infused delivery rates.

The most dramatic effects for UT-15 by either mode of administrationwere on cardiac output and the “lung variables.” U44069 caused cardiacoutput to decrease from the baseline as depicted in FIG. 15. AerosolUT-15 had no effect on cardiac output. Intravenous UT-15 caused adose-response increase in cardiac output, which was significant at 500and 100 ng per kg per min. At 1000 ng per kg per min, aerosolized UT-15delivery was significantly different from the intravenously infusedUT-15.

FIG. 16 graphically demonstrates the overall effects of intravenous andaerosol delivery of UT-15 on pulmonary vascular resistance duringU44069. It shows that pulmonary vascular resistance, while beingsignificantly attenuated by both intravascularly infused and aerosolizedUT-15, was more affected by aerosolized UT-15. In particular, U44069caused a dramatic increase in PVR, which was significantly attenuated at500 and 1000 ng per kg per min for intravenously infused UT-15.Aerosolized UT-15 caused PVR to decrease such that there was nosignificant difference for any of the three delivery rates relative tothe baseline PVR. Interestingly, the time at which intravenous andaerosol UT-15 began to attenuate the increase in PVR were very similarly(4-5 minutes), whereas the off response for aerosolized UT-15 was muchlonger than intravenous UT-15 (43 vs. 12 minutes).

FIG. 18 shows that although intravascular UT-15 caused PPA to decreasesignificantly from the UT44069 value, this decrease matched by adecrease in PLA. Therefore, the pulmonary vascular driving pressure(PPA-PLA) was unchanged.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changed and modifications can be made therein withoutdeparting from the spirit and scope of the invention.

All references cited herein are incorporated by reference to the sameextent as if each was incorporated by reference individually.

What is claimed is:
 1. A method of delivering to a mammal in needthereof a therapeutically effective amount of a benzindene prostaglandincomprising administering to the mammal by inhalation a formulationcomprising droplets measuring less than 10 micrometers in diameter,wherein said droplets comprise a therapeutically effective amount of thebenzindene prostaglandin.
 2. The method of claim 1, wherein said theaerosolized form comprises droplets less than 10 micrometers indiameter, said droplets comprising said benzindene prostaglandin is9-deoxy-2′,9-alpha-methano-3-oxa-4,5,6-trinor3,7-(1′3′-interphenylene)-13,14-dihydro-prostaglandin F₁ in a suitable pharmacologically-acceptableliquid carrier.
 3. The method of claim 1, wherein the mammal is a human.4. The method of claim 1, wherein the formulation comprises a sustainedrelease form of the benzindene prostaglandin.
 5. The method of claim 1,wherein the administering of benzindene prostaglandin has no effect onheart rate.
 6. A method of delivering to a mammal in need thereof atherapeutically effective amount of a benzindene prostaglandincomprising administering to the mammal by inhalation a powderformulation comprising particles measuring less than 10 micrometers indiameter, wherein said particles comprise a therapeutically effectiveamount of the benzindene prostaglandin.
 7. The method of claim 6,wherein the benzindene prostaglandin is UT-15.
 8. The method of claim 6,wherein the mammal is a human.
 9. The method of claim 6, wherein theformulation comprises a sustained release form of the benzindeneprostaglandin.
 10. The method of claim 6, wherein the administering ofthe benzindene prostaglandin has no effect on heart rate.